CZ253899A3 - Antibodies that bind to the laminin domain for nidogen, a method of making such antibodies, and their use - Google Patents

Abstract

Monoclonal and polyclonal antibodies are disclosed as well as parts thereof which bind specifically to the nidogen-binding domain of laminin, as well as a process for producing the same and their use as medicaments, as diagnostic agents for detecting laminin isoforms and as model substances for developing and evaluating substances that influence the nidogen-laminin interaction. The disclosed antibodies or their parts bind preferably to the gamma 1 III 4-domain of laminin, in particular in the highly preserved area of loops a and c, and can inhibit the association of laminin and nidogen.

Description

The present invention relates to monoclonal and polyclonal antibodies and parts thereof that specifically bind to the nidogen binding domain of laminin, to a method for their preparation, and also to their use as a medicament, a diagnostic agent for detecting laminin isoforms, and to their use as a model substance for the development and evaluation of other substances that affect the nidogen-laminin interaction. The antibodies of the invention or parts thereof preferably bind to the γΐ III-4 domain of laminin, in particular to the major nidogen binding site in the highly conserved region of loops a and c, and can thus inhibit the association of laminin and nidogen.

State of the art

The association of laminin (a glycoprotein of 800 kDa) and nidogen (a glycoprotein of 160 kDa) is considered to be a crucial biomolecular mechanism in the synthesis and stabilization of the basement membrane (Mayer, U., Timpl, R. (1994) in: Extracellular Matrix Assembly and Structure (Ed. : P.D.· ,

Yurchenco et al.) pp. 389-416, Academie Press, Orlando, FL). Due to the ability of nidogen to form tertiary complexes with all basic components of the basement membrane, such as laminin isoforms containing γΐ (for nomenclature see: Burgeson, R.E. et al. (1994) Matrix Biology 14:209 - 21 1 ), collagen IV, perlecan and fibulin, or any of their association structures, it takes on the function of a linker that connects different macrostructures to each other, organizes them in space and stabilizes them (Fox, J.W. et al. (1991) EMBO J. 10:3137 3146; Aumailley, M. et al. (1993) Kidney Int. 43:7 - 12). Important information about the central function of the laminin-nidogen interaction in the synthesis of a functional basement membrane can be provided by antibody experiments

The described anti-laminin polyclonal antibodies were obtained by immunization with rabbit laminin P1 or were obtained as a recombinant fragment of laminin γΐ III 3-5 and purified using an affinity matrix of laminin P1 or chromatography on laminin γΐ III-3-5. In inhibition assays they showed* complete inhibition of laminin-nidogen association. This is based on the fact that antibodies whose binding region lies in the vicinity of the binding site of laminin for nidogen sterically block the access of nidogen to laminin (Mayer, U.

12: 1879-1885). In embryonic organ cultures, the described antibodies also inhibited the genesis of renal tubules and also the formation of pulmonary alveoli. Both ontogenetic processes are dependent on unperturbed new synthesis of the basement membrane (Ekblom, P. et al., 1994, Development 120: 2003-2014, Ekblom, P., 1993 in: Molecular and Cellular aspects of Basement Membranes ( (ed. Rohrbach, D.H., Timpl, R.),, pp. 359-383, Academic Press, San Diego, CA).

The binding domain of laminin for nidogen was unambiguously localized, sequence crystallographic, and NMREMBO J. 12: ,1879-1885).

characterized in terms of its spatial structure (X-ray structure) (Mayer, U. et al. (1993)

It exists in the form of the so-called LE module (laminin-type epidermal growth factor - like) of the short arm of the yl chain of laminin in the yl III 4 domain. LE modules are structural motifs composed of 50 to 60 amino acids that form a complex folded *99

9 9

999 999

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9 · · · • 999 9 · · • 9 9 9 9 9

9 9 9 9

999 999 99 9 structure with 4 disulfide bridges analogous to epidermal growth factor (Bairoch, A. (1995) Nomenclature of extracellular domains. The SWISS-PROT Protein sequence data bank. release 310; Engel, J. (1989) FEBS Letters 251:_l-7).

High-affinity binding of nidogen to the complement domain of laminin has been demonstrated for laminin P1 from mouse EHS tumor, laminin-2 and laminin-4 from human placenta, and laminin from Drosophila. The reason for such cross-species binding affinity is the extremely high degree of amino acid sequence identity of the γΐ III-4 domain of laminin in the studied biological species. This identity is 97% for human and mouse and surprisingly 61% for human and Drosophila when the entire module is considered. If only the limited region of loops a to c, which contains the essential binding site for nidogen, is considered, then the degree of identity increases to 100% and in the second case to 75% (Pikkarinen, T. et al. (1987) J. Biol.- Chem. 263: 6751-6758; Chi, H.-C. and Hui, C.-F. (1989) J. Biol. Chem. 264: 1543-1550).

In addition to the fact that nidogen binding is dependent on an intact three-dimensional structure, it is possible to identify certain stretches of sequence that are located in the S-S stabilizing loops a and c of the γΐ III 4 domain. Five essential amino acids have been identified here: four are located within a 7-amino acid stretch in the a loop, and one tyrosine side chain in the c loop (Poschl, E. et al. (1994) EMBO J. 13: 3741-3747; Mayer, U. et al. (1993) EMBO J. 12: 1879-1885;-Poschl, E. et al. (1996) EMBO J.' 15: 5154-5159).

Synthetic peptides that can be derived from the corresponding regions of the laminin γΐ III 4 domain have the ability to completely inhibit laminin-nidogen binding in special binding assays (US patent 5,493,008). Even though these synthetic peptides show an activity in inhibition assays that is 400x to 1000x weaker than the activity of

44 ► 4 4 · » 4 4 4 · · * ·

44« 444 •4 4444 intact laminin P1 or laminin γΐ III-3-5 (Poschi, E. et al. (1994) EMBOJ. 13: 3741-3747; US 5,493,008). The laminin-nidogen interaction is (Mayer, U. et al. (1993) EMBO J. 12: 1879-1885) influenced by a strong conformational component. The weaker inhibitory effect of synthetic peptides is · explainable, since peptides in aqueous solution adopt a myriad of different conformations, and therefore only a certain percentage of the peptide occurs in biologically active conformations.

The use of such peptides as drugs is therefore severely limited due to their conformational flexibility, as well as their instability towards proteases, as well as their poor bioavailability and pharmacodynamics, (Milner-White, E. J. (1989) Trends .Pharmacol. Sci. 10: 70-74; Hrubý, V.J. (1994) in: Peptides,, Proc. Thirteenth American Peptide Symposium; (Ed. : Hodges, R.S. Smith, J.A.) P. 3-17; ESCOM: Leiden, Netherlands).

•Antibodies that specifically bind to the laminin nidogen binding domain, thereby competitively inhibiting the association between laminin and nidogen at low concentrations, are more suitable as drugs for the treatment of certain diseases due to their higher affinity and avidity, greater stability, and good pharmacokinetics. These antibodies can also be used as diagnostic or auxiliary tools in biological and pharmacological models for the development and evaluation of agents affecting the laminin-nidogen interaction.

The antibodies produced so far, anti-laminin P1 or anti-laminin γΐ III-3-5, were only able to partially inhibit binding. Moreover, they did not recognize the binding site for nidogen on the γΐ chain of laminin directly, but inhibited laminin-nidogen binding by spherical action (Mayer, U. et al. (1993) EMBO J.

12: 1879 - 1885). The binding domain of the laminin chain γΐ for nidogen is extremely highly conserved between species. In addition, » · · · ··« ··· • ··· ·« ··«· laminin is an extracellular protein which, as an integral part of the basement membrane and also as a circulating component of serum (see EP 0 696 597 A2), comes into contact with the immune system. Based on the ability of the immune system to distinguish self from non-self, it would follow that each immunized biological species would recognize the highly conserved immunizing antigen as self and would not form antibodies. The formation of specific antibodies is therefore not to be expected. This generally accepted view is confirmed by the fact that no antibodies against the laminin binding domain for nidogen have been obtained so far when immunizing rabbits with laminin P1 and laminin γΐ III-3-5 (Mayer, U. et al. (1993) EMBO J. 12: 1879-1885). The monoclonal antibodies described above, which bind to an imprecisely defined epitope lying outside the laminin binding domain for nidogen, are only of very limited use as drugs, diagnostic agents, or model substances for the development and evaluation of other substances that affect the nidogen-laminin interaction, or not at all: Spherical inhibition depends on the spatial arrangement of the inhibitor, so that for pharmacological reasons the use of parts of these antibodies as drugs is hardly possible. Another limitation for use in diagnostic tests is the possibility of an unwanted analyte.

cross-reactivity of these antibodies

The essence of the invention

The aim of the present invention is to provide antibodies that specifically bind to laminin for nidogen, i.e. directly recognize the nidogen binding domain on the γΐ chain of laminin, and which are intended for use as a drug, diagnostic agent for the detection of • ···· ·· 9 · ’ • ·«· · « • · · · « »» ♦··* ·· · » · · · ·*· · ·· »« · ··· ··

99 laminin isoforms, and as a model substance for, development and evaluation of substances affecting the laminin/nidogen interaction.

The solution according to the present invention is the antibodies described herein, the method of their preparation and their use.

The antibodies according to the present invention are characterized in that they bind to the laminin binding domain for nidogen, namely the γΐ ΙΞΙ 4 domain, preferably a highly conserved section of the a-loop, or loops a and c, of the γΐ III 4 domain of laminin. Particularly preferably, the antibodies according to the present invention bind, with respect to the epitope, in a conformation-dependent manner (the laminin binding site for nidogen recognizes it in its native conformation, cf. Example 6), namely either directly or in an overlapping manner, to the highly conserved sections of the a-loop, or loops a and c. The invention particularly includes antibodies or parts thereof which bind to at least one of the peptides listed in Table 1. The present invention relates to both polyclonal and monoclonal antibodies.

Chimeric antibodies, antibodies.

The oligospecific laminin-nidogen antibodies of the present invention are preferably humanized, bi- or poly-specific. Particularly preferably, the binding by the antibodies of the present invention is inhibited competitively or partially competitively (cf. Example 7).

Table 1

Amino acid sequence of peptides used for immunization

1) DNIDPNAVGNL

2) DNIDPNAVGNLKCIYNTAGFYCDR -(form with S~S bridge) ·· can be obtained «··<

00* ·Φ

0 0 0 • 00 000

I

The antibodies of the present invention are useful for the immunization of immunocompetent vertebrates, such as rabbits, mice, sheep, goats, guinea pigs, rats or chickens, with laminin, laminin P1, laminin γΐ III-3-5, laminin γΐ III-4, and in particular peptides that contain a substantial binding site for nidogen, but not the entire amino acid sequence of the γΐ III-4 domain of laminin, particularly preferred as immunization antigens are either one or both of the peptides listed in Table 1.

In the case of immunization with laminin, or laminin P1, antibodies are identified using laminin γΐ III-3-5 and/or laminin γΐ III-4, and finally tested for competitive or partially competitive inhibition of laminin-nidogen binding.

In the case of immunization with laminin γ1 III-3-5, antibodies are identified through laminin and/or laminin P1 and are finally tested for competitive or partially competitive inhibition of laminin-nidogen binding.

It is particularly preferred to use laminin yl III 4 or one or more peptides according to Table 1 as the immunization antigen. Antibodies obtained by immunization with such an immunization antigen are preferably identified with laminin yl and/or laminin P1. The antibodies thus identified are then tested for competitive or partially competitive inhibition of laminin-nidogen binding.

In addition to polyclonal antibodies, it is also possible to obtain monoclonal antibodies (MAKs) by preparing hybridoma cells producing MAKs. The antibodies can also be obtained in purified form by purifying the antibodies of the present invention from antibody-containing material, such as antiserum from immunized animals, culture supernatants, hybridoma cells, ascites or cells, using • β · · · 9 9·· 9

9999 9 9 9 9 99 9 • 9 9 · 9 9 9 9 999999 • 9 9 9 9 9 9 9

9 9 9 9 9 9 9 9 9 9 9 9 affinity chromatography, preferably on laminin and/or laminin PI as affinity matrix.

The antibodies of the present invention or portions thereof can inhibit the laminin-nidogen interaction and as a general term also include corresponding chimeras, humanized antibodies, bi- or oligospecific antibodies and antibody analogs, which will be described in more detail elsewhere.

The present invention also relates to animal, plant and prokaryotic cells and cell lines which produce antibodies or antibody parts according to the invention, preferably e.g. hybridoma DSMACC 2327, which was deposited on 27 October ¹⁹⁹⁷ in the German Collection of Microorganisms and Cell Cultures, Ltd. (DSMZ, Marscheroder Weg 1b, D-38124 Braunschweig, Germany) in accordance with the Budapest Treaty. The present invention also relates to monoclonal antibodies which are produced by said hybridoma deposited under No. DSMACC 2327.

The present invention further provides a method for preparing the antibodies described above, wherein immunocompetent vertebrates, such as rabbits, mice, sheep, goats, guinea pigs, rats or chickens, are immunized with laminin, laminin P1, laminin γΐ III-3-5 and/or laminin γΐ III 4, preferably with a peptide that does not contain the entire amino acid sequence of the γΐ III 4 domain, and particularly preferably with one or both of the peptides in Table 1. The term peptides is understood to mean both oligopeptides and polypeptides, as well as proteins and protein fragments. The peptides used as immunizing antigens are preferably bound to a carrier such as a protein, e.g. ovalbumin, albumin or hemocyanin, or a polymer, e.g. polyethylene glycol, polyacrylamide or poly-D-glutamine-D-lysine.

• · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ··

In the case of immunization with laminin, or laminin P1, antibodies are identified using laminin γΐ III-3-5 and/or laminin γΐ III-4, and finally tested for competitive or partially competitive inhibition of laminin-nidogen binding.

In the case of immunization with laminin γΐ III-3-5, antibodies are identified through laminin and/or laminin P1 and are finally tested for competitive or partially competitive inhibition of laminin-nidogen binding.

Preferably, in the method of the present invention, laminin γΐ III 4 or either or both of the peptides from Table 1 is used as the immunizing antigen, which is preferably bound to a carrier.

Antibodies prepared by immunization with laminin γΐ III 4 or either or both of the peptides from Table 1 are identified by laminin and/or laminin P1, and are finally preferably tested for competitive or partially competitive inhibition of laminin-nidogen binding.

The method of the present invention comprises the preparation of monoclonal antibody (MAK) hybridoma cells. It has been shown that the antibodies of the present invention or portions thereof can be purified from antibody-containing material, such as antisera from immunized animals, supernatant from hybridoma cell cultures, ascites or cells, by affinity chromatography, preferably on laminin and/or laminin P1 as the affinity matrix.

The antibodies of the present invention or their parts can be used in many ways, e.g. as a drug, as a diagnostic agent, as an aid in biological and pharmacological models for the development and evaluation of substances affecting the laminin-nidogen interaction, as a model substance for evaluating the spatial structure of the contact zone of complement optionally also producing it is advantageous • · » ·

Furthermore, the present invention relates to the use of one or more antibodies according to the present invention or parts thereof for the manufacture of a medicament for the treatment of diseases characterized by increased or unwanted synthesis of the basement membrane, in particular fibrosis, especially alcoholic liver fibrosis, pulmonary fibrosis, and also all forms of late complications of diabetes, which are accompanied by thickening of the basement membrane, in particular in the kidneys, eye and vascular system, also arteriosclerosis and all diseases in which angiogenesis contributes to the deterioration of the clinical picture, such as cancer diseases, diabetic retinopathy, and diseases with a strong inflammatory component such as rheumatoid arthritis, osteoarthritis, vasculitis, hemangioma and psoriasis.

• production of a diagnostic agent for the detection of laminin isoforms containing γΐ in biological samples, e.g. body fluids such as blood, serum, plasma, urine, saliva or cerebrospinal fluid, as well as in tissues. The term diagnostic agent is understood to mean various forms of heterogeneous and homogeneous immunoassays, test kits for immunohistochemistry, as well as reagents for in vivo diagnostics such as e.g. immunoscintigraphy. The agents containing antibodies according to the present invention or parts thereof may be either alone or may be in the form of a diagnostic kit together with other auxiliary reagents such as buffers, washing solutions, calibration solutions and other auxiliary means such as e.g. cuvettes.

In the following part of the description, the invention will be explained in more detail and examples of embodiments of the invention will be given in detail.

Surprisingly, antibodies against the highly conserved amino acid sequences of the laminin nidogen binding domain were prepared using the peptides listed in Table 1, which do not form any folded structure such as LE modules. For this purpose, the peptides listed in Table 1 were carbodiimide-linked to ovalbumin, and these conjugates were used to immunize rabbits. The development of the titer of specific antibodies against laminin P1 and laminin γΐ III-3-5 was monitored by enzyme immunoassay.

Polyclonal antibodies with the desired specificity were purified by affinity chromatography with a matrix bound to laminin P1 from human placenta and finally by gel filtration chromatography. The purification and characterization of laminin from human placenta and its use for immunization of mice and for obtaining hybridomas synthesizing anti-laminin-P1 antibodies have been described in EP 0 696 597 A2.

Antibodies obtained by affinity chromatography to laminin P1 show binding specificity for laminin P1 from human placenta, laminin P1 from mouse (EHS tumor) and laminin from rat (yolk sac). The antibodies recognize not only specific sequences but also biologically active conformations of the laminin binding domain for nidogen. They are capable of completely inhibiting laminin-nidogen binding. The antibodies according to the present invention can also be obtained by immunizing other immunocompetent vertebrates, e.g. rabbits, mice, sheep, goats, guinea pigs, rats or chickens, namely with laminin, laminin • «

P1, laminin γΐ III-3-5, laminin γΐ III 4, and especially peptides that contain a substantial nidogen binding site, but not the entire amino acid sequence of the γΐ III 4 domain of laminin, particularly preferred as immunizing antigens are either one or both of the peptides listed in Table 1. The polyclonal antibodies of the present invention can then be purified from the serum of immunized animals.

For the preparation of the corresponding monoclonal antibodies, it is possible to use generally known methods (see e.g. Harlow, E. and Lané, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor), in which immune cells of an immunized animal, e.g. a mouse, are fused with myeloma cells, thereby preparing hybridoma cells producing monoclonal antibodies (MAK), and finally certain clones are isolated. The selection of the desired MAK-producing clones is carried out using specific test methods. The affinity of the antibodies secreted into the immunizing antigen, to the carrier, is tested by enzyme transfer on cultured immunizing laminin.

binding media of the antigen, to native or recombinant or to fragments thereof, preferably by immunoassay, radioimmunoassay or Western blot Another possible selection criterion is the ability of the antibodies to disrupt the laminin-nidogen binding. This can be tested, for example, by an inhibition assay, which is described in detail in the examples. Hybridomas that produce MAK specifically binding to the laminin binding domain for nidogen are then cloned. They are then permanently available for MAK production. Depending on the desired purpose of use, it may be advantageous to prepare only parts of the antibodies, e.g. F(ab) ₂ , Fab' or Fab fragments. These can be prepared, for example, by enzymatic cleavage known to the skilled person (see, for example, Harlow, E. & Lané, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor).

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The antibodies of the present invention and portions thereof may be modified, e.g., by labeling them with a radioactive isotope or paramagnetic compound for use in in vivo diagnostics, or by linking them to a pharmaceutically active agent, thereby preparing an even more effective drug.

The following examples serve to explain in more detail individual aspects of the present invention.

Examples of embodiments of the invention

SDS-electrophoresis, Western blotting and BCA protein assays were performed according to standard procedures or according to the manufacturers' instructions. Unless otherwise stated, chemicals used were obtained from Merck (Darmstadt), Sigma (Munich) or Riedel de Haen (Seelze).

Example 1

Peptide synthesis

The solid phase peptide synthesis was carried out on an ABI 433 peptide synthesizer, with 148 mg (0.1 mmol) of amide-tethered FMOC-PAM resin. After the synthesis was complete, the resin had gained weight to 513 mg. The resin was then washed with a solution of 10 mL of trifluoroacetic acid and 365 µl of triethylsilane at room temperature for 2 hours. After filtering off the resin residues, the solution was rotated under vacuum, precipitated with 50 mL of 10% acetic acid (AcOH) and lyophilized. This gave 145 mg (54% yield) of crude peptide. The crude peptide was then dissolved in 3 mL of 80% AcOH and this solution was added dropwise to a rapidly stirred solution containing 0.006 mmol of iodine and 0.006 mmol of sodium acetate in 55 mL of 80% AcOH. "After 5 • ·· · • ·· minutes, the reaction was terminated by adding 0.1 N ascorbic acid solution. The solution was then concentrated to a volume of 2 ml and applied to a Sephadex® G25 column activated with 0.1 M AcOH. The isolated peptide was then chromatographically purified to perfection using reverse-phase HPLC.

Yield: 36 mg of peptide DNIDPNAVGNLKCIYNTAGFYCCDR-NH ₂ (13.5% of theory).

The structure was confirmed by mass spectroscopy (molecular weight 2673 Da) and amino acid analysis.

The peptide was also prepared in an analogous manner

DNIDPNAVGNL-NH ₂ .'

Yield: -268 mg peptide (67% of theory), molecular weight 1140 Da.

Example 2,

Peptide binding to ovalbumin mg ovalbumin (Sigma A-2512) was dissolved in 1 ml phosphate buffer, pH 7.4 and 200 μl of an aqueous solution of 7 mg sodium salt of N-hydroxysulfosuccinate (Fluka 56485) and 300 μl of an aqueous solution of 100 mg of 1-ethyl-3-(3-diaminopropyl)-carbodiimide-HCl (Sigma E-6383) were added. After 5 minutes, a solution of the peptide (30 mg of the corresponding peptide in 1 ml of 10 mM Na-phosphate buffer, pH 7.4) was added. The conjugation reaction was carried out for 16 hours at room temperature in the dark. After this time, the solution was centrifuged to remove turbidity. Unreacted chemicals and salts were removed by chromatography on a NAP-25 column (Pharmacia). The ovalbumin-peptide conjugate was thus transferred to PBS+0.04% Tween 20. The yield was 50-55 mg of conjugate.

····

Conjugate-1:

ovalbumin-DNIDPNAVGNL

Conjugate-2:

ovalbumin-DNIDPNAVGNLKCIYNTAGFYCDR (form with S-S bridge)

Example 3

Immunization of rabbits

Mixed-breed rabbits weighing about 3 kg were immunized with an ovalbumin-peptide conjugate. For this purpose, 1 mg of the corresponding conjugate was dissolved in 0.5 ml of phosphate-buffered saline (PBS) and then supplemented with an equal volume of Freund's adjuvant and carefully emulsified. 1 ml of the emulsion thus obtained was injected intradermally into the rabbits, namely one-fifth of the volume at five different sites near the lymph nodes. After 21 and 53 days, booster injections with half the concentration of the antigen emulsion (the antigen was emulsified with Freund's adjuvant) were administered.

Example 4

Antibody titer development in four immunized rabbits

The development of specific antibody titers in 4 animals was monitored in more detail by enzyme immunoassay using microplates with wells coated with laminin P1 or laminin γΐ III-3-5. The corresponding animal sera were designated K2, K3, K4 and K905. Sera K3 and K4 were obtained after immunization with conjugate 1, sera K2 and K905 after immunization with conjugate 2.

, All rabbits developed an immune response very quickly, ···· • ·

This may indicate the existence of multiple populations of antibodies in polyclonal serum that bind with different affinity to the nidogen binding motif, or its conformation, in laminin P1 and laminin γΐ III-3-5.

Example 5

Affinity purification of antibodies

For a more detailed characterization, the specific antibodies were separated from the remaining immunoglobulins in the animal serum, other serum components and antibodies directed against ovalbumin. For this purpose, affinity chromatography with an affinity matrix with laminin P1 was used. Fractogel® EMD Azlacton 650 S (Merck, Darmstadt) was used as the carrier (gel matrix). 0.3 g of this material was incubated in 6 ml PBS, 1M Na ₂ SO ₃ , pH 7.4 for 15 minutes before conjugation, and the excess solution was then decanted. During the incubation, the material swelled to a volume of 1 ml and the matrix could thus be used directly for the covalent binding of the desired ligand.

3.4 g of human laminin P1 was dissolved in 2 ml of 0.2M ammonium carbonate, pH 8.0, and incubated with 1 ml of activated carrier (see above) at 4°C overnight (>16 hours). Finally, the gel material was rinsed with 0.2M ammonium carbonate, pH 8.0. The remaining active groups of the carrier material were blocked by 24-hour incubation with 5 ml of ···· ···♦ • · · · • * · ···«· ·«· · · · · 9 9 9 9

9 9 · 9 9 9·· 99 9 ·'' 9 9 9 9

9 99 9 9 9 99 99 nm) flow rate Then followed

0.2M glycine, pH 8.0, at 4°C. Matrix preparation for affinity binding was completed by three wash cycles consisting of alternating incubation in PBS, _{1M Na2SO3} , pH 7.4, 0, 1M sodium acetate, pH 4.0, and 0.2M glycine, pH 8.0. The matrix was loaded onto a Pharmacia HR 5/5 column and equilibrated with PBS/0.04% Tween 20.

For purification of laminin Pl-binding antibodies from l

animal serum, the serum was diluted 1:2 with PBS with 0.04% Tween 20 and loaded onto the column at a rate of 2 ml/min. The column was then washed with buffer until the UV signal (220 of the monitor reached the baseline value again, elution of bound antibodies, and .sice so that the loading buffer was exchanged for 0.1M glycine/HCl with pH 2.7.

Analysis of the flow-through solution and the eluate was ^performed using an enzyme immunoassay with immobilized laminin γ1 II-3-5.

Antibodies were purified from the sera of all four animals by the affinity purification method described above. The yield was on average 0.3 mg per 50 ml of serum (unfortunately, in the case of serum K2, the yield was only 0.1 mg). However, the majority of antibodies in all sera (>80%) did not bind to laminin P1, probably due to slow binding kinetics or to the extraordinary affinity of the peptide sequences used for immunization.'

Affinity chromatography on a laminin P1 buffer resulted in the selective purification of a stable and fast binding antibody variant (the target antibody) from serum.

Example 6

Binding specificity of affinity purified antibodies

It has been shown by Western blotting that affinity purified antibodies retain their binding specificity for laminin PI and that various preparations react more strongly with unreduced laminin PI than with a linear fragment of laminin PI separable by reduction of disulfide bonds (Gerl, M et al., 1991, Eur. J. Biochem.

202: 167-174). This indicates a conformation-dependent component of the antibody binding specificity. Furthermore, it was shown that the four prepared antibodies differed in their binding specificity. Both antibody preparations K3 and K4 obtained by immunization with conjugate 1 recognized identical laminin P1 bands after non-reducing SDS (sodium dodecyl sulfate) gel electrophoresis. Both antibody preparations K2 and K905 (i.e. anti-conjugate 2) showed an identical reaction pattern, but recognized fewer laminin P1 bands than the antibody preparations anti-conjugate 1. In an immunochemical test with laminin P1 bands separated by reducing SDS electrophoresis, it was apparent that the individual antibody preparations have different binding preferences for fragments containing laminin γΐ III 4.

Example 7

Inhibition assay: Inhibition of laminin-nidogen binding by affinity purified antibody

The inhibitory activity of an affinity purified antibody can be identified using a coated tube assay, which measures the binding of radiolabeled nidogen to the wall of a laminin PI (from human placenta) coated tube in the presence of antibodies.

Radioactive labeling of nidogen with ¹²⁵ iodine

Recombinantly obtained human nidogen (35 μς, description of clones, cultivation and purification conditions - see Meyer, U. et al., 1995, Eur. J. Biochem. 227: 681-686) was dissolved in • · · · ··· ·· ·· ·· • · · · · · · « · 9 9 99 9 9 9 99 9

999 99 9 999999

19 9 9 999 999 9 9 9 9 99 9 .99 250 μΐ PBS, and then 0.405 was added mCi ( = 15MBq) Nal solution ₍ U5 I) ( = 1.55 μΐ, Nordion Europe), 10 μΐ 0.5M Na-phosphate, pH 7.4, and 40 μg chloramine T (sodium salt amide N-chloro-4- toluenesulfonic acid, Merck) in 100 μΐ 0.05M Na-phosphate, pH 7.4. The reaction took 60 seconds at temperature rooms and was

stopped by adding a solution of 40 μρ sodium metabisulfite (Riedel de Haen) in 100 μΐ 0.05M Na-phosphate, pH 7.4. This took place within 30 seconds at most, then 900 μΐ 1% BSA. (Sigma) in PBS was added. Separation of free radioactivity and excess salts was carried out by gel filtration chromatography with a PD 10 column (Pharmacia). The eluted iodinated nidogen in PBS was combined and then diluted with a solution of ·1%BSA/0.05 Na-phosphate/0.01%NaAzide, pH 7.4, so that the final concentration was 50 ng/ml.

Coating of reaction tubes

Reaction tubes (Greiner, 75x12, no. 115061) were coated with a solution of laminin P1, 4 μς/ηχΐ in carbonate buffer (0.159 g Na ₂ CO ₃ , 0.293 NaHCO ₃ , 0.02g NaN ₃ vil distilled water), 20 μg/ml BSA (bovine serum albumin, Serva), pH 9.2, overnight at 4°C. Free binding sites were then blocked by 2-hour incubation with 0.5' ml 0.5% BSA in PBS/0.04% Tween 20.

Inhibition test with laminin mimetic structures (gradual inhibition)

In a reaction tube, 200 μΐ of iodinated nidogen (10 ng, i.e. about 40,000 cpm) and 200 μΐ of inhibitor (e.g., a peptide derived from the yl III 4 domain of laminin) or standard (laminin yl III-35) were shaken at room temperature. Both inhibitor and standard were dissolved in PBS/0.04% Tween 20. After incubation for 3 hours, 150 μΐ of this mixture was transferred to a coated tube and incubated for another • 99 9

9 9

9 9

9 9

9999 • 9 · 9 9 9 • 999 99 9 hours at room temperature. The solution was then decanted, the tube was rinsed twice with 1 ml PBS/0.04% Tween 20, and the bound radioactivity (nidogen) was measured in a gamma counter. The amount of bound nidogen in the inhibitor solution was compared to nidogen without the addition of inhibitor.

Inhibition test with nidogenmimetic structures (gradual inhibition)

In reaction tubes coated with laminin P1, 150 μΐ of inhibitor (i.e., an antibody binding to the γΐ III 4 domain of laminin or a peptide derived from the nidogen sequence) or standard (which is recombinant nidogen) were shaken for 1 hour. Both inhibitor and standard were dissolved in PBS/0.04% Tween 20. After aspirating the sample, 150 μΐ of iodinated nidogen (10 ng, i.e., about 40,000 cpm) was added for 2 hours to displace the bound inhibitor. Finally, the solution was decanted, the tube was rinsed twice with 1 ml PBS/0.04% Tween 20, and the bound radioactivity (nidogen) was measured in a gamma counter. The amount of bound radioactive nidogen was compared with the inhibitor or the standard concentration, respectively.

Inhibition test (simultaneous inhibition)

In this variant of the assay, no pre-incubation was performed. In several replicates, 75 μΐ of iodinated nidogen (10 ng) and 75 μΐ of inhibitor (or standard) were pipetted into a coated tube and incubated for 2 hours at room temperature, the rest being carried out analogously to the variant of the assay with gradual inhibition.

Results

From 10 independently performed tests with the standard laminin γΐ III-3-5, an IC50% value of 0.22nM was calculated with a standard deviation of

9999'

99

9999 with a standard deviation of ± 15%. The IC ₅₀ % value is defined as the concentration of the substance required to achieve 50% inhibition of the binding of nidogen to laminin P1. The test 'described in document' US 5,493,008 (equilibrium inhibition test) gave a corresponding IC ₅₀ % value of 0.05 nM with a standard deviation of ± 52%.

Table 2 shows the IC50% values for the antibody preparations K3, K905, K1.2, K2.2 and K3.2 as well as for the various free peptides. Antibody preparations K1.2, K2.2 and K3.2 originate from the second immunization of rabbits with the new conjugate 2 and were purified in a comparable manner. The results demonstrate the reproducibility of the entire procedure.

Table 2

Inhibition of laminin-nidogen binding by antibodies of the invention

Inhibitor K3 K905 Cl.2 K2.2 K3.2 Peptide(1)* Peptide(2)* ₅₀ % ₅₀ % IC _50% IC ₅₀ % IC ₅₀ % IC ₅₀ %' ₅₀ % nM nM nM nM nM nM nM gradual** inhibition 72 150 ' 500 80 350 60,000 20,000 with imultaneous inhibition** 110 - 600 80 500 60,000 20,000

* amino acid sequence see table 1 ** test type 'In document US 5 493 008, IC50% values between 22 nM and 1000 nM were reported for inhibitory peptides derived from the binding domain for nidogen. These values could not be achieved with the selected test due to the drastically shortened incubation time; the DNIDPNAVGNL peptide had an _IC50% value of 60 000 nM in the test described here.

Example 8

Characterization of binding kinetics using the BIAcore® system

Using the BIAcore® system from Pharmacia Biosensor, it is possible to monitor biospecific interactions in on-line mode. The measurement principle is based on an optical phenomenon (surface plasmon resonance), which is influenced by the mass bound to the gold film. In simple terms, it is a miniaturized affinity chromatography on the surface of a gold sensor. The amount of specifically bound ligand can be displayed in the form of a resonance signal (Chaiken, I. et al., 1992, Anal. Biochem. 201: 197-201, Karlsson, R. et al., 1992, in: Structure of Antigens, (ed. van Regenmortel), pp. 127-148, CRC Press, Boca Raton, Fl). Laminin PI was immobilized on the sensor chip at a concentration of 200 µg/ml in 10 mM Na-acetate, pH 4.0, according to the manufacturer's instructions. This created a matrix with 4000 RU of bound laminin PI. A double pulse of 4 μl of 100 mM HCl can be performed to regenerate the affinity matrix.

At a flow rate of 2 μΐ/min in HBS buffer (10 mM HEPES, 3.4 mM EDTA, 150 mM NaCl, 0.005% BIA-surfactant P20, pH 7.4), nidogen (20 μg/ml) bound to laminin P1 with parabolic saturation kinetics and affinity purified antibody preparations K905 and K3 with linear dependence on antigen. The reason that no transition to the equilibrium state was observed even after 1400 seconds may be the fact that the specific sequence of laminin P1, which is responsible for nidogen binding, was well accessible to antibodies. The antibody bound to this sequence regardless of whether it was in the biologically active conformation or in another conformation. This is evidenced by • ···

9 · · • 9 99 · · • · 9 9 '9 • · · ·

999 99 *9 999**

99 • 9 · · · • 9 9 9 9 • 9 999 999 • 9 9 • 9 9 99 observation that nidogen already showed a clear transition to the saturation phase after binding 600 RU. Apparently, not all immobilized laminin Pl molecules have a structure recognizable by nidogen, so that the theoretical saturation value corresponding to the level of 2500 RU will not be reached. However, both antibodies reached it with a longer contact time. It was striking that the K905 sample had to be used in a tenfold higher concentration (320 μg/ml) than K3 (33 μg/ml) to achieve the same binding rate. The binding of K905 to laminin Pl was, on the other hand, more stable than K3, since the dissociation rate of K905 (determined from the HBS buffer exchange time: 1500 seconds) was significantly slower than that of the K3 preparation. ,

These findings explain the differences between K3 and K905 found in inhibition assays:

• Antibody preparation K3 inhibits laminin-nidogen binding better than K905, as it is characterized by faster association kinetics.

• The K3 antibody preparation inhibits even when simultaneously incubated with nidogen in a tube coated with laminin P1, as it has good association kinetics at a concentration comparable to that of nidogen.

• The antibody preparation K905 inhibits only in the variant of the test with gradual inhibition, as it is characterized by a slower dissociation rate and therefore it is no longer possible to displace it well by the later added nidogen. In the current competition for binding sites, the preparation K905, due to its slower association kinetics, succumbs to nidogen.

·· ·· • · · · · • · · · · · • · «·· ··· • · · · • ·· · * ···· • 9 9 9 · • ··· * · • · · · · · • · · · «····· »· • 4 ···<»

Example 9

Demonstration of binding specificity of affinity purified antibody preparations K3 and K905 using Western blotting

Western blot analysis was performed to examine the interaction of K3 and K905 antibody preparations with conjugate-2 and ovalbumin. Antigens were electrophoretically separated on 4 to 12% NuPAGE™ gels (Novex™, San Diego, CA) with MOPS buffer according to the manufacturer's instructions (Novex™) and then transferred to a nitrocellulose membrane using NuPAGE™ transfer buffer (Novex™). After blocking the free binding sites on the membrane with 1 µg/ml polyvinyl alcohol (1 minute), the antigens were incubated with the test antibodies. Detection of bound antibodies was then performed using anti-rabbit IgG antibody to which alkaline phosphatase was covalently attached.

The affinity purified antibodies were shown to bind exclusively to the peptide, while interaction with the carrier protein ovalbumin was not detectable. No interaction was observed with the blue-stained marker See Blue standard (Novex™).

Furthermore, both K3 and K905 were shown to react with laminin and laminin derivatives from various biological species (laminin from human placenta, laminin from rat yolk sac (Calbiochem), laminin P1 from human placenta, laminin from EHS mouse tumor, recombinantly prepared mouse laminin γΐ HI-3-5). This is a consequence of the binding of the antibody to a conserved sequence within the binding domain for nidogen. None of the antibody preparations showed cross-reactivity with human nidogen or human collagen type IV. This is a prerequisite for the use of the antibodies according to the present invention as nidogen antagonists.

• 0 · •00 00· •Example 10

Preparation of monoclonal antibodies

For the preparation of monoclonal antibodies, selected vertebrates, preferably mice or rats, were immunized in a standard manner with, for example, laminin, laminin P1, laminin γΐ III3-5, laminin γΐ III 4 or the conjugates mentioned in Example 2. In the case of a specific immune reaction of the antisera, hybridomas producing monoclonal antibodies (MAKs) were then obtained in a standard manner.

The screening of the desired antibodies, or the corresponding hybridoma clones, was carried out, among other things, by a binding assay (e.g. dot blot assay or western blot with laminin yl III-3-5 or laminin yl III 4), and in particular by means of the inhibition assays described in Example 7. The clones thus selected represent a source for the synthesis of large quantities of antibodies according to the present invention.

For the purification of the desired antibodies, it is possible to use conventional methods such as binding to protein G or protein A. It is preferable to use affinity chromatography with a laminin P1 column. This purification step allows, as in the case of the polyclonal antibodies described above, the selection and selective enrichment of monoclonal antibodies with the best binding constant.

An example of an antibody according to the present invention is a monoclonal antibody (MAK) which is produced by the cell clone A6/2/4, which was deposited as accession no. DSMACC 2327 on October 27, 1997 in the German Collection of Microorganisms and Cell Cultures, Ltd. (DSMZ, Marscheroder Weg lb, D-38124 Braunschweig, Germany) in accordance with the Budapest Treaty.

This hybridoma originates from the immunization of mice with laminin P1, which was isolated from human placenta. Antigen purification

4 4 4 4 4 • 4 4 4 4 4 · 4 · 4 · • · 4 4 4.4 .4

4 44 4 4 4 4

4 4 4 4 4 4 *4 4 4

6 and the preparation of the hybridoma are described in EP 0 696 597 A2. The A6/2/4 antibody was identified on the basis of its binding properties to laminin γΐ III-3-5 and laminin γΐ III 4. It is a monoclonal antibody of the IgM subtype that can be purified by gel chromatography. The binding of this antibody to laminin Pΐ is extremely strong due to the determined polyvalency. For this reason and also due to the size of IgM antibodies, it is not possible to achieve elution from an affinity column with laminin Pΐ. The strong binding to laminin Pΐ is also reflected in the inhibition test (simultaneous variant). The monoclonal antibody A6/2/4 is able to inhibit the laminin-nidogen association with an IC ₅₀ value of 30 nM.

Due to the strong conformational dependence of binding, it is not possible to clearly delineate the binding epitope in the laminin γΐ III 4 domain (laminin nidogen binding domain). The fact that peptides derived from the laminin nidogen binding sequence partially inhibit the interaction of antibodies with laminin P1 (75 to 80%) means that the binding epitope of the A6/2/4 antibody overlaps with nidogen.

The following section describes the preparation of inhibitory monoclonal antibodies, which, like the polyclonal antibodies of the present invention described above, were prepared using peptide-ovalbumin conjugates.

SJL/J mice were immunized by subcutaneous injection of 50 μg of peptide 2-ovalbumin conjugate (conjugate 2, see above) in the presence of Freund's complete adjuvant. After 4 and 8 weeks, the immune response was boosted by another subcutaneous injection of 25 μg of conjugate 2 in Freund's complete adjuvant, followed by a 7-week wait. Three days before the fusion, the immune • · » ·

I 9

9 • · · · response further enhanced by intraperitoneal injection of additional μς of conjugate 2.

For fusion, mice were sacrificed and spleen cells were harvested. In the presence of polyethylene glycol, the cells were fused with the myeloma cell line P3X63AG8.653. Hybrids (spleen cells x P3X63AG8.653) were selected by culturing the fusion mixture in a medium with hypoxanthine, aminopetrine and thymidine for 3 weeks. In order to obtain a stable line, the selected cell clones were subcloned several times. The resulting cell colonies were tested for antibody production using various binding immunoassays. The resulting cell lines E79/1/6 and E82/1/10 were selected based on the screening strategy described below.

Tests used to characterize and identify specific monoclonal antibodies

Immunization of SJL/J mice with conjugate 2 resulted in the generation of an exceptionally large number of antibody-producing hybridoma clones. Antibodies from the culture medium showed a strong immune reaction with laminin γΐ III-3-5, laminin Pΐ and ovalbumin. ,

In order to find clones that produce monoclonal antibodies against nidogen with the native structure of the nidogen binding domain, an appropriate screening had to be performed. The main consideration of this method is the focus on the binding of the antibody to laminin P1 and laminin γΐ III-3-5. At the same time, due to subcloning, care must be taken that the reaction with the carrier protein, ovalbumin, is negligible and antibodies that exclusively recognize ovalbumin are eliminated.

Tables 3 and 4 show the results obtained with the two clones thus identified (E79 and E82).

• ·· ·

Table 3

Determination of E79 binding by ELISA

Laminin yl 111-3-5 coating: 2.5 pg/ml Laminin Pl coating: 2.0 pg/ml Coated with ovalbumin 20 pg/ml Hybridom E79, undiluted culture medium 1.33 0.68 1.63 E79/1/6, 1st cloning undiluted culture medium 2, 03 0.16 0.27 E79/1/6 purified antibody 2.5 pg/ml 0.56 0.33 0.05

Table 4

Determination of E82 binding by ELISA

Laminin coating yl III-3-5: 2.5 pg/ml Laminin Pl coating: 2.0 pg/ml Ovalbumin coating 20 pg/ml Hybridom E82, undiluted culture medium 1.77 1, 48 2.33 E82/1/10, 1st cloning undiluted culture medium 1.32 0.26 0.05 E79/1/6 purified antibody 6.4 pg/ml 1.55 0.5 0.18

By cloning the cells, it was apparently possible to separate the cells that showed an immune reaction with ovalbumin. At the same time, a cell clone could be isolated that produces an antibody that shows binding to laminin γΐ III-3-5 and laminin Pl. This fact, as well as the fact that immunization with a peptide derived from the laminin binding domain for nidogen was successful, demonstrates that the antibody formed binds to the laminin binding motif for nidogen with a native structure.

Direct evidence of binding specificity was provided by a simultaneous variant of the inhibition assay (see above), where prepared antibodies directly compete with iodinated nidogen for binding to the nidogen binding motif of intact laminin (laminin from mouse EHS tumor, Chemicon, no. CC095). The antibody (subtype IgG2a) produced by cell clone E79/1/6 inhibits laminin-nidogen association with an IC50 value of 19 nM, the antibody (subtype IgG1) produced by cell clone E82/1/10 inhibits laminin-nidogen association with an IC50 value of 190 nM.

Claims (39)

1. An antibody that binds to the nidogen γΐ III-4 binding domain of laminin, or a portion of such an antibody. 2. An antibody or part thereof according to claim 1, which binds to a highly conserved region of loop a, or loops a and c, of the nidogen binding domain of laminin γΐ III-4 or in the immediate vicinity of this loop. 3. The antibody or part thereof according to claim 2, which, with respect to the epitope, binds directly or in an overlapping manner, depending on the conformation, to a highly conserved region of the a-loop, or of the a- and c-loops. 4. An antibody or part thereof according to one or more of the preceding claims 1 to 3, which binds to at least one of the peptides listed in Table 1. The antibody according to one or more of the preceding claims 1 to 4, which is a polyclonal antibody. The antibody according to one or more of the preceding claims 1 to 4', which is a monoclonal antibody. 7. The antibody of claim 6, which is a chimeric, humanized, bi- or oligospecific antibody. An antibody according to one or more of the preceding claims 1 to 7, which competitively or partially competitively inhibits laminin-nidogen binding. 0 0 0 0 •00 000 9. , An antibody obtained by immunizing an immunocompetent vertebrate with laminin or laminin P10 as an immunizing antigen, and then identifying the antibody with laminin γΐ III-3-5 and/or laminin γΐ III-4 and testing for competitive or partially competitive inhibition of laminin-nidogen binding. 10. An antibody obtained by immunizing an immunocompetent vertebrate with laminin γΐ III-3-5 as an immunizing antigen, and then identifying the antibody with laminin and/or laminin P1 and testing for competitive or partially competitive inhibition of laminin-nidogen binding. 11. An antibody obtained by immunizing an immunocompetent vertebrate with an immunizing antigen that is laminin γΐ III-4 and/or peptides that contain a substantial portion of the nidogen binding site, but not the entire amino acid sequence of the γΐ III-4 domain of laminin. 12. The antibody according to claim - 11, which is obtained by using laminin γΐ III-4 as the immunizing antigen. 13. The antibody of claim 11, which is obtained by using as an immunizing antigen either one or both of the peptides listed in Table 1. The antibody according to one or more of claims 11 to 13, which is identified by laminin and/or laminin P1. 4 4 4· • 44 • · 4 · · 4 4 4 4 4 4 4 4 4 4 444 4 4 β 444 444 4 4 4 4 · · · ······ ·4 4 «· ·· 15. An antibody according to one or more of claims 11 to 14, which has been tested for competitive or partially competitive inhibition of laminin-nidogen binding. 16. The antibody of any one of claims 9 to 15, which is produced by hybridoma cells producing a monoclonal antibody. 17. The antibody of any one of claims 9 to 16, which is obtained by purifying the antibody-containing material by affinity chromatography. 18. The antibody of claim 17, wherein the affinity chromatography is performed on a laminin or laminin P1 matrix. 19. A cell or cell line that produces an antibody according to one or more of claims 1 to 8 or a portion of said antibody. 20. Hybridoma DSMACC2327. 21. An antibody that is produced by the hybridoma DSMACC2327. 22. A method for producing an antibody according to any one of claims 1 to 8, characterized in that an immunocompetent vertebrate is immunized with laminin or laminin P1, the antibody is identified with laminin γΐ III-3-5 and/or laminin γΐ III-4, and then tested for competitive or partially competitive inhibition of laminin-nidogen binding. ···· • · « · • · ♦ · * · · « · · « · · · · · 4 · 4 · • 4 · · · · 4 · ···««· • · · · · · 4 · !·»«<· »· · 4 4 44 23. A method of producing an antibody according to any one of claims 1 to 8, characterized in that an immunocompetent vertebrate is immunized with laminin γΐ III-3-5, the antibody is identified with laminin and/or laminin P1, and then tested for competitive or partially competitive inhibition of laminin-nidogen binding. 24. A method of producing an antibody according to any one of claims 1 to 8, characterized in that an immunocompetent vertebrate is immunized with laminin γΐ III-4 and/or peptides that contain a substantial portion of the nidogen binding site, but not the entire amino acid sequence of the γΐ III 4 domain of laminin. 25. The method according to claim 24, characterized in that laminin γΐ III-4 is used as the immunizing antigen. 26. The method of claim 24, wherein either one or both of the peptides listed in Table 1 are used as the immunizing antigen. 27. The method of claim 26, wherein the immunizing antigen is bound to a carrier. 28. The method according to one or more of claims 24 to 27, characterized in that the antibody is identified by means of laminin and/or laminin P1. 29. The method according to one or more of claims 24 to 28, characterized in that the antibody is tested for competitive or partially competitive inhibition of laminin-nidogen binding. ft · · · · · • ft· 3 4 ' • · · · · ······ · · · « 30. Method according to one or several claims 22 up to 2 9 characteristic with by that, that will prepare hybridoma cells producing monoclonal antibody. 31. Method according to one or several claims 22 up to 30 characteristic with by that, that the antibody cleans from the material containing antibody using affinity chromatography • 32. The method of claim 31 v y z listen moving by that, that an affinity test will be performed chromatography on affinity matrix laminin and/or laminin Pl. 33. An antibody or portion thereof according to any one of claims 1 to 18 or 21 for use as a medicament. 34. A medicament characterized in that it contains one or more antibodies or parts thereof according to at least one of claims 1 to 18 and 21. 35. Use of one or more antibodies or parts thereof according to at least one of claims 1 to 18 and 21 for the manufacture of a medicament for the treatment of diseases characterized by increased or unwanted basement membrane synthesis. 36. Use according to claim 35, wherein the disease is a form of late complications of diabetes accompanied by thickening of the basement membrane, a form of arteriosclerosis, fibrosis or a disease in which angiogenesis contributes to the worsening of the clinical picture. • · · · · · • flflflfl • · · fl • flflfl · • flflfl • flfl • flfl flflfl flfl flfl • flflfl • flfl · • flfl flflfl • · 37. Use according to claim 35 or 36 for the manufacture of a medicament for the treatment of diabetic retinopathy, alcoholic liver fibrosis, pulmonary fibrosis, cancer, diabetic nephropathy or diseases with a strong inflammatory component such as rheumatoid arthritis, osteoarthritis, vasculitis, hemangioma and psoriasis. 38. An antibody or portion thereof according to any one of claims 1 to 18 or 21 for use as a diagnostic agent. 39. A diagnostic agent characterized in that it contains one or more antibodies or parts thereof according to any one of claims 1 to 18 and 21. 40. Use of one or more antibodies or parts thereof according to at least one of claims 1 to 18 and 21 for the production of a diagnostic agent for detecting γΐ-containing laminin isoforms in biological samples, body fluids or tissues. 41. Use of one or more antibodies or parts thereof according to at least one of claims 1 to 18 and 21 as an aid in biological and pharmacological models for the development and evaluation of substances influencing the laminin-nidogen interaction. 42. Use according to claim 41 in a biological or pharmacological model for the development and evaluation of substances influencing the laminin-nidogen interaction.